1. Field of the Invention
The invention relates to a method for exciting and determining a luminescence of an analyte which is located in contact with a surface of a waveguiding layer of a layer waveguide. In a case of which, a luminescence is generated in a non-evanescent way in the volume of the analyte which leads to an optical measuring device from the immediate proximity on the surface of the waveguiding layer luminescence radiation which has penetrated into the waveguiding layer of the layer waveguide, preferably via at least one outcoupling element for the luminescence radiation. Then, the optical measuring device measures the luminescence light, for example, optoelectronically. The invention also relates to a measuring device for carrying out the method, and to a sensor platform.
2. Description of the Related Art
In affinity sensor technology, biochemical detection elements are immobilized on a waveguide surface either directly or via an adhesion promoter layer, for the purpose of specifically detecting an analyte in a sample, which can consist of a complex mixture of substances, and for the purpose of binding the analyte molecules on the surface of the wave guide, in the region of the depth of penetration of the evanescent field. In order to detect the, analyte, the sample in solution is brought into contact with detection elements immobilized on the waveguide surface, either in stop and flow or in throughflow.
Planar waveguides have recently been developed in the field, in particular, of biochemical analysis, for the purpose of generating and detecting evanescently excited radiation. In the evanescent field, there is generated a luminescence in contact with an analyte sample, for example, fluorescence, whose measurement permits a qualitative or quantitative determination of substances even in very low concentrations. The evanescently excited radiation emerging isotropically into space is determined optoelectronically by means of suitable measuring devices such as, for example, photodiodes, photomultipliers or CCD cameras. This method is disclosed, for example, in WO 95/33197. It is also possible for the fraction of the evanescently excited radiation coupled back into the waveguide to be coupled out via a diffractive optical element, for example, a grating, and measured. This method is described, for example, in WO 95133198. For the purpose of simultaneously or sequentially carrying out multiple measurements, arrays have become known in which at least two waveguides are arranged on a sensor platform which are driven separately with the aid of excitation lightxe2x80x94see, for example, WO 96135940.
The known measuring methods place high requirements on the positioning accuracy of the excitation light with reference to the incoupling elements in order to achieve an adequate light incoupling and thus sensitivity. The use of adjusting components is therefore mandatory, and this complicates the technical design and is evident, in particular, in the case of the construction of arrays.
In addition, one is limited to the use of essentially coherent light in order to coordinate the positioning with the constants of the incoupling elements and such as, for example, diffractive gratings.
For classic, highly multimodal waveguides, such as, for example, multimode capillaries, multimode glass fibers or multimode glass platelets, the problem of high positioning requirements for coupling in the excitation light can be circumvented by applying the so-called xe2x80x9cluminescence concentration principlexe2x80x9d as described, for example, in Sensors and Actuators B 38 to 39 (1997), pages 96 to 102 and pages 300 to 304. However, here use is made of optical waveguides which comprise the substrate itself (without an additional more highly refractive layer), which is located in an environment with a low refractive index, and in the case of which waveguides a geometrical shape permits total reflection. It is described that the luminescence light from the emission sources applied to the substrate surface, such as, a polymer membrane with an embedded indicator dye, is collected over a large solid angle and then guided in the glass substrate to a detector located on an end face of the waveguide. Such indicator dyes are typically used in high, for example, millimolar, concentrations. Such thick glass substrates used as multimodal waveguides are not suitable for measuring very low detection concentrations.
It has now been found, surprisingly, that even in the case of optical layer waveguides comprising a transparent substrate and a highly refractive waveguiding layer it is possible to apply the principle of the xe2x80x9cluminescence collectorxe2x80x9d, and thus the problems associated with the coupling in of the excitation light are completely avoided when the excitation radiation is directed without the use of incoupling elements at least partially directly onto the volume of the analyte sample in order to generate the luminescence, for example, in a reflected-light or transmission arrangement. The luminescence radiation generated in the analyte sample in the immediate proximity of the surface of the waveguiding layer is, surprisingly, coupled into the waveguiding layer to a measurable extent and can, for example, be detected optoelectronically at the end face of optical fibers or planar waveguides, or via outcoupling elements in the case of planar waveguides. Luminescence generated in the further analyte volume is, surprisingly, virtually not coupled into the waveguiding layer, as a result of which interfering luminescence radiation generated in the a analyte is excluded, and a virtually background-free measurement is permitted which has high spatial selectivity, high efficiency and high sensitivity.
Layer structures composed of a transparent substrate such as, for example, glass, quartz or plastics such as polycarbonate, with a lower refractive index than the highly refractive waveguiding layer, applied to the surface, within a refractive index of for example at least 1.8 are as layer waveguides within the scope of the invention. The thickness of the waveguiding layer is preferably selected such that it can guide only a single or only a few (for example, up to 1) discrete modes of light of a specific wavelength. The layer waveguides are denoted below as waveguides for short.
It has been found, surprisingly, that not only luminescence excited by optical radiation, but even luminescence generated by other mechanisms such as, for example, chemiluminescence, triboluminescence, bioluminescence or electroluminescence, can be measured optoelectronically with the aid of layer waveguides, and this makes available a new method for highly sensitive determination of such luminescence radiation.
Direct irradiation of the analyte sample located in contact with the waveguide surface offers the following advantages, for example:
very sensitive detection with tithe aid of a configuration corresponding to conventional epifluorescence excitation;
use of coherent or noncoherent radiation sources, since the luminescence is not generated by the evanescent field of excitation radiation guided in a waveguiding layer, but luminescence radiation generated in the immediate proximity of the surface of the waveguiding layer of a waveguide is measured;
distinction between volumetric luminescence and luminescence radiation generated in the immediate optical proximity, which permits a measurement in turbid analyte samples such as, for example, blood, serum or reaction mixtures;
low requirements on the positioning accuracy of the excitation light;
low technical outlay in the use of sensor platforms with at least two separate waveguiding regions (sensor fields) for simultaneous measurements;
virtually background-free detection by means of a detection position completely separate in space from the site of excitation;
technically simple implementation of array formats such as, for example, a microtiter plate format, with adaptation to standardized sizes;
economic, cost-effective production even of compact forms of sensor systems, since requirements are placed on optomechanical adjusting devices;
use of cost-effective, freely selectable and commercial light sources, the wavelength region being set, if appropriate, by filters,
use of excitation light with a wavelength of  less than 450 nm, and possibility of excitation even with the aid of UV light;
use of sensor platforms with open cutouts in the nl to xcexcl regions for holding samples;
use of layer waveguides; and
low energy density of the excitation radiation, accompanied by gentle treatment of the analyte samples.